This invention pertains to disulfide-linked conjugates of therapeutic agents containing at least one thioamide group with thiol-containing polymers, so as to provide a controlled release pharmaceutical composition for administration to animals for the prophylaxis or treatment of various conditions or diseases.
Derivatives of polymers such as polyethylene glycol (PEG) containing thiol (xe2x80x94SH) groups may be used as a controlled release carrier for therapeutic agents with thiol groups, by administering the polymer to which the therapeutic agent is linked by a disulfide bridge. Reduction of the disulfide group by endogenous reducing agents results in the release of the therapeutic agent (Huang et al.[10]; Woghiren et al.[19]). Furthermore, the therapeutic agent linked to the polymer may be in an inactive, or prodrug form, which when released becomes active. The inclusion of various targeting agents which also have been conjugated to the same polymer to target the therapeutic agent to particular sites within the body or to enhance cellular uptake have been described.
Appended PEG chains may provide the favorable pharmacologic properties of protection of the underlying protein from immune surveillance and proteolytic enzymes, in addition to a lower rate of clearance from the bloodstream (Davis et al., 1981). Furthermore, based on the properties provided by the PEG portion of the conjugate (Davis et al., 1981), conjugates of therapeutic agents as prodrugs with polymers provides certain advantages such as reduction in possible toxicity, since biological activity of a large bolus of that drug would not appear immediately upon administration to the patient. Thus, the biological activity might be present at a relatively constant, therapeutic level in the bloodstream over an extended time period due to two opposing actions, the conversion of inactive prodrug to active drug and the clearance of active drug from the body.
While therapeutic agents which have a thiol group, or may be derivatized to have one without loss of activity, are suitable for the above process, numerous other compounds without such groups cannot be bound to thiol-containing polymers following standard procedures to produce a controlled release composition. This is particularly true for compounds with a thioamide group. It is toward the development of a controlled release delivery system for therapeutic agents with thioamide groups that the present application is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
In its broadest aspect, the present invention is directed to a pharmaceutical composition which is a disulfide-linked conjugate between at least one therapeutic agent comprising prior to conjugation a thioamide moiety, and at least one polymer comprising prior to conjugation at least one thiol group. The polymeric portion of the polymer which comprises prior to conjugation at least one thiol group may be a homopolymer or a copolymer, and may be by way of non-limiting example, polyethylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide, copolymers of ethylene/maleic anhydride copolymer, polylactide/polyglycolide copolymers, polyaminoacids, copolymer of polyethylene glycol and an amino acid, or polypropylene oxide/ethylene oxide copolymers. The polymer may also be a branched polymer or a dendrimer, i.e., a multi-branched polymer.
In a preferred embodiment, the polymer is a polyethylene glycol polymer (PEG), for example, of a molecular weight of from about 300 to about 30,000 Da, and preferably, from about 600 to about 5,000 Da. The PEG has functional groups or may be derivatized to bear functional groups to which a compound providing a free thiol group may be attached. The polymer comprising at least one thiol group may have from one to about ten thiol groups per polymer; preferably from one to about three thiol groups per polymer. The thiol group on the polymer may be sterically hindered.
The polymer comprising at least one thiol group may be prepared from, for example, xcex1,xcfx89-diamino-polyethylene glycol and thiomalic acid; xcex1,xcfx89-dihydroxy-polyethylene glycol and thiomalic acid; xcex1,xcfx89-dicarboxy-polyethylene glycol and cysteamine; xcex1,xcfx89-dicarboxy-poly(ethylene glycol) and 1-amino-2-methyl-2-propanethiol; or xcex1,xcfx89-dicarboxy-PEG subunits and lysine, wherein carboxy groups on the lysines are derivatized to form thiol groups. The selection of the thiol compound providing the disulfide link to the thioamide-containing compounds and the covalent link to the polymer may be selected from a number of compounds containing a thiol group and a reactive group which may be attached to a polymer.
The therapeutic agent comprising prior to conjugation a thioamide moiety may be an agent that contains such a thioamide group in its active form, or a therapeutic agent which is modified to contain a thioamide group. For example, therapeutic agents with thioamide groups include UC781; R82150; HBY097; troviridine; S2720; UC38and 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-fluoro-4-thiothymidine. However, the invention is not so limiting. Furthermore, other compounds with thioamide-like groups of similar reactivity to thioamide-containing compounds as described herein are likewise suitable as compositions as described herein. Such compounds include but are not limited to thioureas and thiourethans.
In a further aspect of the invention, the polymer may additionally have a functional group, which may be derivatized with a compound such as but not limited to a cell uptake enhancer or a tissue targeting agent.
The composition of the present invention may include a second therapeutic agent, or a second and a third therapeutic agent. This may be achieved by preparing polymers conjugated to each therapeutic agent separately, and then mixing these polymers to provide a composition with more than one therapeutic agent. In another embodiment, a single polymer to which at least two thiol groups is attached may be derivatized with a mixture of therapeutic agents. The relative amounts of the different agents conjugated to the polymer may be selected to correspond with the therapeutic effectiveness of each compound. The therapeutic agents conjugated to the polymer of the invention are released in vivo under reducing conditions. The in-vivo half life of the therapeutic agent in the composition may be increased compared with that of the therapeutic agent alone in vivo. Furthermore, the therapeutic agent may be therapeutically inactive or weakly active in the composition. The water solubility of the therapeutic agent may be increased in said composition compared to its inherent water solubility.
In another broad aspect, the invention is directed to a pharmaceutical composition which is a disulfide-linked conjugate between at least one therapeutic agent comprising prior to conjugation a thioamide moiety, a bifunctional compound comprising prior to conjugation at least one thiol group, and at least one polymer attached to one or more of the bifunctional compounds. The polymer may be a homopolymer or a copolymer, and may be by way of non-limiting example, poly(ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide, copolymers of ethylene/maleic anhydride copolymer, polylactide/polyglycolide copolymers, polyaminoacids, copolymer of polyethylene glycol and an amino acid, or polypropylene oxide/ethylene oxide copolymers. The polymer may also be a branched polymer or a dendrimer, i.e., a multi-branched polymer. For linkage to the bifunctional compound, the polymer may have one or more similar or different functional groups such as an amino or carboxy group, which may be cross-linked to the functional group on the bifunctional compound with a cross-linking agent, or the polymer may be an activated polymer, such as but not limited to polyethylene glycol bis(imidazoyl carbonyl), which is capable of reacting with an amino group. The bifunctional compound comprising prior to conjugation at least one thiol group may for example comprise a thiol group and an amino group, such as but not limited to cysteamine or 1-amino-2-methyl-2-propanethiol.
In another broad aspect of the present invention, methods for preparing a composition comprising a disulfide-linked conjugate of at least one therapeutic agent comprising prior to conjugation a thioamide moiety with at least one polymer comprising prior to conjugation at least one thiol group are described. The conjugate may be prepared by following the steps of: (A) providing at least one therapeutic agent comprising a thioamide moiety or modified to have a thioamide moiety; (B) providing at least one polymer comprising at least one thiol group; (C) reacting the at least one therapeutic agent comprising a thioamide moiety under oxidizing conditions to form a disulfide cross-linked dimer of the therapeutic agent comprising a thioamide moiety; (D) reacting the disulfide-linked dimer with the polymer comprising at least one thiol group, under conditions in which a disulfide exchange reaction occurs to form a disulfide-linked conjugate between the therapeutic agent comprising a thioamide moiety, and the at least one polymer comprising at least one thiol group; and (E) isolating the disulfide-linked conjugate.
The thiol-containing polymer may have a thiol-containing moiety thereon, which may be prepared by any of a number of methods. By way of non-limiting example, a compound with a thiol group and another functional group, for example, an amino group, may be covalently coupled to a polymer with carboxylic acid moieties, for example, 1-amino-2-methyl-2-propanethiol or cysteamine may be conjugated to a PEG polymer with carboxylic acid moieties, using a carbodiimide reagent. By way of another example, thiol-containing compounds containing a carboxylic acid moiety such as thiomalic acid may be conjugated to a PEG bearing amino moieties using a carbodiimide. In the previous examples, the thiol group of 1-amino-2-methyl-2-propanethiol is sterically hindered, while that of cysteamine is less so.
The polymer comprising at least one thiol group may be prepared from, for example, xcex1,xcfx89-diamino-poly(ethylene glycol) and thiomalic acid; xcex1,xcfx89-dihydroxy-poly(ethylene glycol) and thiomalic acid; xcex1,xcfx89-dicarboxy-poly(ethylene glycol) and cysteamine; xcex1,xcfx89-dicarboxy-poly(ethylene glycol) and 1-amino-2-methyl-2-propanethiol; or xcex1,xcfx89-dicarboxy-PEG subunits and lysine, wherein carboxy groups on the lysine are derivatized to form thiol groups. The selection of the thiol compound providing the disulfide link to the thioamide-containing compounds and the covalent link to the polymer may be selected from a number of compounds containing a thiol group and a reactive group which may be attached to a polymer.
In a further aspect of the invention, methods for preparing a composition comprising a disulfide-linked conjugate between at least one therapeutic agent comprising prior to conjugation a thioamide moiety, a bifunctional compound comprising prior to conjugation at least one thiol group, and at least one polymer attached to one or more bifunctional compounds are described. In one embodiment, a disulfide exchange-produced heterodimer is prepared between the thioamide-containing compound and a bifunctional compound comprising at least one thiol group and an amino group, thus forming a disulfide-linked conjugate comprising the therapeutic agent and the functional (amino) group. Subsequently, the functional group of the heterodimer is covalently linked to the polymer, for example, using a cross-linking reagent to cross-link the functional group of the bifunctional compound and a functional group on the polymer, or by use of an activated polymer capable of reacting directly with the functional group on the bifunctional compound. In yet a further embodiment, the bifunctional group comprising a thiol group is first conjugated to the polymer, for example by any of the foregoing methods, leaving at least one free thiol group, and subsequently, a homodimer of the oxidized therapeutic agent is reacted under disulfide exchange conditions with the polymer to produce the desired conjugate. In this fashion, additional control over the selection of sterically hindered thiol groups is provided to tailor the release characteristics of the therapeutic agent to the particular condition to be treated or prevented, and the target organ, tissue or cells.
The polymer portion of the polymer comprising at least one thiol group and the bifunctional compound are as described hereinabove.
The therapeutic agents in the present invention have a thioamide group, whether present in or introduced synthetically into the agent. For example, the agent may be UC781; R82150; HBY097; troviridine; S2720; thiouridine; UC38 and 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-fluoro-4-thiothymidine. Many other therapeutic agents, for various uses, are embraced herein. Furthermore, a therapeutic agent may be prepared or chemically modified to provide a therapeutically active analog having a thioamide group.
The oxidizing conditions to form the dimer of the thioamide-containing therapeutic agent comprises reaction in the presence of an oxidizing agent which may include, but is not limited to, molecular oxygen, hydrogen peroxide, and molecular iodine. The subsequent disulfide exchange reaction may be carried out under conditions which promote the reaction, for example, in a degassed nonaqueous solvent, such as a 1:1 mixture of dimethylformamide and dichloromethane. The invention is not so limiting to these conditions and any suitable conditions may be employed to achieve the preparation of the desired conjugate.
Furthermore, any therapeutic agent having a thioamide-like group that may be oxidized to form a dimer and then attachable via a disulfide exchange reaction to a thiol-containing polymer via a disulfide bond is suitable for use herein. Such moieties such as but not limited to thioureas and thiourethans are included herein.
The polymer comprising at least one thiol group may additionally have a functional group, such as an amino or carboxyl group, and by way of non-limiting examples, the additional functional group is optionally derivatized with a cell uptake enhancer or a tissue targeting agent. The polymer may also be a branched polymer or a dendrimer, i.e., a multi-branched polymer.
A pharmaceutical composition of the present invention prepared as described above may have a second therapeutic agent or a second and third therapeutic agent. The preparation step may include a mixture of disulfide-linked dimers of more than one agent, which during the disulfide exchange reaction become conjugated to the polymer. The relative efficiency of disulfide exchange and/or the relative concentration for each dimer may be used to provide conditions such that the resulting polymer has the therapeutic agents present at the desired ratio. In another embodiment, polymers conjugated separately to particular therapeutic agents following the process described above are mixed at the desired ratio before administration to the patient.
The therapeutic agent is of the composition released in vivo under reducing conditions. The in-vivo half life of said therapeutic agent in the composition may be increased compared with that of the therapeutic agent alone in vivo. Furthermore, the therapeutic agent may be therapeutically inactive or weakly active in the composition. The water solubility of the therapeutic agent may be increased in said composition compared to its inherent water solubility. In a further embodiment, the disulfide-linked agent polymer conjugate may be entrapped in a matrix providing a controlled release depot.
In another aspect of the present invention, a method is provided for the controlled release in an animal of at least one therapeutic agent having a thioamide moiety comprising administering to the animal a composition comprising a pharmaceutical composition as described hereinabove. The composition may comprise a second therapeutic agent, or a second and a third therapeutic agent. The therapeutic agent may be released in vivo under reducing conditions.
The in-vivo half life of said therapeutic agent in the animal is increased compared with that of the therapeutic agent alone in said animal. The therapeutic agent may be therapeutically inactive or weakly active in said composition.
In another aspect of the present invention, a method is provided for the controlled release in an animal of a therapeutic agent within a preselected body compartment comprising administering to said animal a composition comprising the pharmaceutical composition as described above, wherein the pharmaceutical composition additionally comprises a targeting agent for targeting said composition to said compartment. Non-limiting examples of targeting agents include an antibody, a cell uptake enhancer, or a tissue targeting agent.
These and other aspects of the present invention will be better appreciated by reference to the following drawing and Detailed Description.